Heterokaryosis as a Cause of Culture Rundown in PenicilliumF. L HAAS, T. A. PUGLISI, A. J. MOSES, AND J. LEIN

Research Division, Bristol Laboratories Inc., Syracuse, New York

Received for publication February 27, 1956

Physiological degeneration of fungus cultures haslong been a problem encountered in industrial fermen-tations. This has been no less true of antibiotic fermen-tations than of the older ones. Degeneration in anti-biotic-producing cultures is usually accompanied by anumber of cultural and biochemical changes, some ofwhich may be correlated in a general way with reductionin antibiotic production by the specific culture. How-ever, a given change is usually an indicator of physio-logical degeneration only in the case of a specific straincultured and fermented under a specific set of condi-tions. For example, Foster et al. (1943) and Foster(1949) find that physiological degeneration usually isaccompanied by reduction in sporulation and advisethat a minimum number of transfers from heavilysporulating isolates should be used as inoculum. On theother hand, Whiffen and Savage (1947) found thatsporulation itself was a factor promoting "penicillinrundown" (the term given by them to physiologicaldegeneration in regard to penicillin production), andthat rundown did not occur over as many as 50 consecu-tive vegetative transfers if all sporulation was pre-vented. In our experiments we find that even spore-to-spore transfers over more than 25 serial transfers willnot promote rundown, provided a rigid system ofselection is available and utilized. Other seeminglyconflicting reports are quite common for penicillin andother antibiotic fermentations.

It is generally thought that changes in the strainleading to physiological degeneration are of geneticorigin arising as gene mutations somewhere in thehistory of the strain. However, the general mechanismby which these changes are disseminated and preservedin the mold cultures and the possible role of hetero-karyosis in physiological degeneration, have not beenconsidered in detail in relation to industrial fermenta-tions.Many genetically controlled changes are scattered

and preserved in most of the Fungi Imperfecti, as wellas in many of those having a perfect stage, by hetero-karyosis. Hyphae of the same or different strains formanastomoses to initiate this process. This is followedby nuclear migration across the anastomoses, subse-quent mixing of nuclear types within the same hyphaeand, finally, adjustment of nuclear numbers of thetwo types within the mycelia to a ratio which affordsthe maximum growth rate under the immediate en-vironmental conditions.

Jinks (1952a, 1952b) found that most Penicilliumstrains isolated from natural sources (soil and decayingvegetable matter) were stable heterokaryons. He wasalso able clearly to demonstrate that such a stableheterokaryon had a growth rate superior to those of itshomokaryotic component strains. Whenever a com-ponent strain attained a growth rate superior to thatof the heterokaryon the heterokaryon would break downand at least one pure homokaryotic strain developedin mixture with the heterokaryon. Thus heterokaryonstability and breakdown appear to follow the growthrate theory of heterokaryosis advanced by Beadle andCoonradt (1944) for Neurospora.

Proof that heterokaryosis occurs in Penicillium, andthe mechanics of nuclear behavior (migration andmixing) at heterokaryon formation, have been presentedby Baker (1944) and by Pontecorvo and Gemmell(1944a, 1944b). The mechanics are the same as givenby Hansen (1938, 1942), Hansen et al. (1932, 1943),Dodge (1942), Dowding and Buller (1940), and Beadleand Coonradt (1944) for a number of other fungi.Heterokaryosis has since been demonstrated in Peni-cillium many times, but to our knowledge the roleplayed by this process in influencing fermentation-production yields has not been presented. Certainresults of Foster et al. (1943) could be interpreted asshowing the effect of heterokaryosis in causing penicillinrundown, but this possibility was not investigated.One of the strains studied by us was Wisconsin 50-

935 (see Campbell and Curtis, 1952, for origin andhistory). Some mutants obtained from this strain wereexcellent for a study of the role of heterokaryosis inpenicillin rundown, since they coupled distinct culturalcharacters with characteristic fermentation yields ofpenicillin. They also proved of value in studying thevariation pattern which has been observed in all of theWisconsin series since Q-176. This paper presents theresults of a series of experiments with these mutantsdesigned to test the effects of heterokaryosis on peni-cillin yields, its involvement in penicillin rundown, andthe origin of different strains observed in the normalpopulation of strain W50-935.

MATERIALS AND METHODS

The following Penicillium strains were used in theexperiments reported: W50-935, strains A, B, C, D,E-1, E-2, H, and 2158. Strain W50-935 was obtainedoriginally as a single-colony isolate from a plating of

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nitrogen-mustard-treated W49-133 (Campbell andCurtis, 1952). A slant of this strain kindly supplied byDr. M. P. Backus of the University of Wisconsin,Department of Botany, served as the origin of ourstocks of this strain. In our hands this strain appearsto be a mixture of types. Several morphologicallydifferent populations are visible when it is plated onlactose-cornsteep medium (LCS), and these can beseparated from each other by single colony isolationfrom this medium. In shake-flask fermentations thisstrain produces approximately 900 Oxford units ofpenicillin per ml of broth under our conditions. StrainsA, B, C, D, E-1, E-2, and H are all isolates establishedfrom a plating of ultraviolet-irradiated W50-935spores. All of these isolates were olive green, heavilysporulated, and seemed to be composed of the samecolony type. In shake-flask fermentations they allproduced approximately 3000 oxford units per ml ofpenicillin. In some experiments, series of substrains wereestablished from these strains by selecting isolatedcolonies, single spores, or single hyphal tips from plat-ings. Strain 2158 was previously obtained at BristolLaboratories' Research Department as a selection froman ultraviolet-irradiated spore suspension of Wisconsinstrain W48-701 (see Backus and Stauffer, 1955, forhistory of W48-701). Soil-tube cultures were made ofeach of the strains, and the same soil tube of eachstrain was used throughout this study to establishprimary cultures. To determine which of the abovestrains would undergo penicillin rundown, serialtransfers of each strain were made on LCS agar slants.Transfers were made by suspending the spores andmycelia from a slant, or spores from a soil tube, in 0.01per cent Wetanol.1 This mass inoculum was used toinoculate fresh agar slants. Slant growth from eachculture was transferred in the same manner to freshslants after 6, 14 and 21 days' growth at room tempera-ture (RT). This procedure was carried out through sixserial transfers with each strain. The spore-myceliasuspension was also used to inoculate triplicate fermen-tation shake flasks at the time of inoculating eachslant. Each shake flask contained 100 ml of LCS liquidmedium. These fermentations were incubated for 8days at 76 F on a rotary shaker operating at 200 rpm.Five-ml aliquots were removed from each flask on the5th, 6th, 7th, and 8th days of fermentation and analyzedfor pH, penicillin production, and population changes.Penicillin assays were run by a modification of the"cylinder-plate" method (Schmidt and Moyer, 1944)on centrifuged broth samples. Population patterns, andchanges in them, were observed by plating series ofserial dilutions of uncentrifuged broths on LCS agar.Triplicate plates were made at each dilution by theglass-rod-spreader technique. These plates were incu-bated at RT for 9 days and then examined for colony

types and percentages of each type calculated. Theshake-flask medium used in most cases was a modifica-tion of the LCS medium of Moyer and Coghill (1946).LCS agar slants were made of the same medium with2.5 per cent Difco agar added. In some experimentsfor observing population changes and heterokaryonbreakdown, honey-peptone (Churchill, 1947) andmodified Czapek-Dox (Jinks, 1952a) media were used.

All inocula from slants, colonies, or fermentationbroths used in experiments reported here were macer-ated for 30 sec. in a Waring blendor before use.Inoculum for small tank fermentors was grown on

LCS agar flats in 10-L wide-mouth bottles. These cul-tures were grown for 7 days at 76 F and then harvestedaseptically in 0.01 per cent Wetanol. Samples of theharvested tank inocula were tested for penicillinproduction in shake-flask fermentations, and forcolony types by the plating technique at the same timethat they were tested in the tank fermentors.

Single-spore isolations were made from most of thecultures with a de Fonbrune micromanipulator, usinga modified Zelle technique (Zelle, 1951). Hyphal tipswere isolated with a Chambers micromanipulator undera dissecting microscope.

Heterokaryon formation between pairs of all strainswas checked microscopically, using the method ofLindegren (1934). In general, this method was notsuccessful because of a decided reluctance of the strainsto form hyphal anastomoses on the solid medium used.

RESULTS AND DISCUSSIONPenicillin Rundown in Isolates of W50-935

During a routine investigation of strain W50-935numbers of isolates were obtained from a screening ofultraviolet-irradiated spores which gave penicillinyields considerably higher than those obtained underthe same fermentation conditions with the parentstrain. These isolates were maintained on LCS agarslants and in soil-tube cultures. Several of them weretested for penicillin rundown by serial transfer ofmass inoculum on LCS agar slants after various periodsof growth. One series of transfers was started from soiltubes and another from slants inoculated with theprimary colony at the time it was isolated from theoriginal plates. At the time of each transfer the slantwas tested in shake-flask fermentations for penicillinproduction and was plated out in serial dilutions onLCS agar for cultural examination. All of the strainstested in this manner exhibited rapid rundown andafter three serial transfers produced only 20 to 50 percent as much penicillin as the original isolate. At thesame time, growth became heavier and the fermentationcycle shorter. The parent W50-935 strain was carriedas a control throughout these experiments and did notrundown or change growth patterns through a total ofnine serial transfers. However, its penicillin production

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was only approximately 25 per cent that of the isolatesbefore they underwent rundown. Platings of the inoculaat each transfer showed that certain cultural changesparalleling pencillin rundown occurred in the cultures.The original isolates and first slants of the isolates re-vealed only a single dark-green, heavily sporulatingcolony type (G-type) whenplated on LCS agar. However,at various stages of transfer, usually after the secondbut sometimes after only one transplanting, a few whitecolonies (W-type) and white-sectored dark-green colo-nies (S-type) would appear in the platings. The W-typestrain is apparently a pure albino type. It sporulatesalmost as well as the G-type and its spores, as well asmycelium, have no pigment. The parent strain shows allthree of these types, among others, when it is plated onLCS agar. In further transfers of the isolates, the per-centages of W- and S-type colonies increased. Withintwo transfers after the first appearance of W- or S-typecolonies the isolate had the same population patternand penicillin production as W50-935. Transfer experi-ments were carried out in three series: one with a 7-daygrowth period, a second with a 12-day growth period,and a third with a 21-day growth period between trans-fers. Rundown was much slower, and cultural changesappeared later in the 7-day-old transfer series. Usually,when 12-day- and 21-day-old slants were used, rundownand cultural variants were pronounced after the firsttransfer. The age at which sectoring occurred, and itsmanner of appearance, remind one of similar sectoringobserved by Churchill (1947) in earlier ancestors ofW50-935. He found that flesh-colored, nonsporulatingvariants of Q-176 would produce heavily sporulatinggreen sectors after 7 days' growth. He further foundthat this sectoring could be prevented indefinitely bytransferring hyphal tips from colonies less than a weekold, or by growing the colonies continuously on freshmedium.Whiffen and Savage (1947) found that penicillin

rundown in Penicillium notatum NRRL 1249 B21 couldbe delayed by any process that decreased the percentageof spores in the inoculum. Using vegetative inoculagrown under conditions that permitted almost nosporulation, they could prevent rundown indefinitely.To determine if rundown in our strains could be con-trolled by similar methods, experiments were run inwhich the cultures were kept in the vegetative state.This was accomplished by serial transfers of shake-flaskgrowth at 24-hr intervals to fresh shake flasks of LCSmedium. At each transfer aliquots were used to inocu-late other shake-flask fermentations for penicillin pro-duction analysis. They were also plated out in serialdilutions on LCS agar for observation of populationchanges. The results showed that rundown and popula-tion changes occurred much earlier under these condi-tions than when 7-day-old slant transfers were made.

Using vegetative transfers, rundowrn usually startedwith the second transfer.

Plating Studies on White, Green and SectoringSubstrains of Strain A

Studies on the colony types appearing in strain Aimmediately before and after the start of rundown werecarried out on LCS agar by serial hyphal tip transfersand by mass inocula platings from single colonies of theG-, W-, and S- types. These studies showed that theW-type was very stable. No changes appeared in thesesubstrains through as many as five transfers. Moreover,when platings were grown for as long as 25 days no sec-toring appeared on any W-type isolate. The G-typewas relatively stable and little or no sectoring or whitecolonies occurred throughout the series of transferswhen they were made at 7-day intervals. In many casessectors would appear in small numbers in platings whenthey were held for longer periods of time; however, a fewG-type substrains were established which would notsector regardless of the length of time grown. Theselatter strains showed no indication of rundown throughas many as five serial transfers of mass inoculum. S-typecolonies were unstable and mass inocula platings alwaysproduced all three colony types, with S-type coloniesaccounting for 75 per cent or more of the population.Hyphal tip transfers from white sectors always pro-duced only W-type colonies, while hyphal tip transfersfrom the green portions of S-type colonies almost al-ways produced S-type colonies. In a few cases, G-typecolonies were produced from hyphal tips taken from thegreen portion. These plating experiments were alsocarried out on honey-peptone agar and modifiedCzapek-Dox agar. On these media S-type colonies didnot appear. Platings of the G-type colonies yield homo-geneous light-green populations, while platings of theW-type colonies yield homogeneous white populations.Mass inocula platings of S-type colonies from LCS agarplates onto honey-peptone or Czapek-Dox agar platesyield only colonies typical of the W- and G-types. How-ever, when the G-type colonies produced by platingS-type colonies on honey-peptone or Czakep-Dox areplated back onto LCS agar they are found to be of twotypes, those yielding only G-type, and those yieldingG-, W-, and S-types. Therefore, while sectoring is pre-vented phenotypically on Czapek-Dox and honey-peptone media the factors responsible for the sectoringon LCS remain present in the mycelium and must be ofa genetic nature. The most logical mechanism whichcould explain this behavior would be heterokaryosis inwhich two types of nuclei are present in the mycelium.This heterokaryon would necessarily remain balancedon Czapek-Dox and honey-peptone media, but wouldbreak down on LCS medium. Such a system would bevery similar to that observed by Jinks (1952b) andRees and Jinks (1952), who found that Penicillium

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TABLE 1. Fermentation and population characteristics of sub-strains isolated from platings of vegetative growth of strain Aafter 8 days' growth in shake flasks

Substrain (Colony Type)* Penicillin Production Population Pattern onLactose-Cornsteep Agar

Oxford units/ml

G-type .............. 2760 G-type colonies 'G-type .............. 2700 G-type coloniesS-type .............. 1980 G-, W-, and S-type

coloniesS-type .............. 1670 G-, W-, and S-type

coloniesW-type .............. 590 W-type coloniesW-type .............. 500 W-type colonies

* G-type = heavily sporulating colony type; S-type =white-sectored, dark-green colony type; and W-type = whitecolonies.

heterokaryons isolated from natural sources were stableonly on media containing large amounts of apple pulpbut broke down on simpler media. Penicillin rundownwas not studied in cultures grown on honey-peptone orCzapek-Dox media, since slants grown on these mediagave very low penicillin yields when used as inocula inour shake-flask fermentation medium.

Effect on Penicillin Production of the G-, S-, and W-typeColonies

Since S- and W-type variants appeared more rapidlyin vegetative liquid cultures than on solid media, it wasprobable that they would also arise in fermentationshake flasks and in the various stages of tank fermenta-tions. If this occurred, penicillin yields would probablybe reduced proportionate to the time of appearance ofthe variants and to the extent of their growth, since W-and S-types were always found to give low penicillinyields. A study of this possibility was carried out usingslants of a G-type isolate of W50-935. The slants of thisstrain (strain A) were inoculated from a soil tube whichhad previously been tested and found to contain ahomogeneous G-type population giving good penicillinyields in shake-flask fermentations. The slants were usedto inoculate shake-flask fermentations which were runfor 8 days. Samples of the shake-flask growth wereplated out in serial dilutions at 2-day intervals, and thefermentation broths were assayed for penicillin potencyon the 6th, 7th, and 8th days of fermentation. Theplatings revealed that S-type colonies started to appearon the 4th and 5th days of fermentation. By the 8thday approximately 40 per cent of the colonies wereS-type, and a few of the W-type were also noted.Whether or not these changes interfered with penicillinproduction in these fermentations could not be deter-mined. Average maximum potencies of 2850 Oxfordunits per ml were attained, and this was about the sameas had been obtained in previous experiments withstrain A. The possibility exists, however, that the yields

would have been higher if such variants could havebeen prevented from appearing at all. The experimentswere repeated using different variations of the LCSmedium. None of these variations had any noticeableeffect on the cultural changes or their time of appear-ance. Single-colony isolations were made of each of thethree colony types appearing in this fermentation. Theseisolates were then used as inocula for similar fermen-tations which were investigated by the same platingand penicillin potency tests. The results of theseexperiments are given in table 1. They show that certainisolates (W- and S-type colonies) taken from the normalfermentations of the G-type strain give fermentationpotencies similar to those of rundown cultures. In thecase of the S-type isolate, platings of 7-day-old fermen-tation flask growth show approximately the same ratiosof G-, S-, and W-type colonies as are found in platingsof rundown cultures. Results of only a few substrainstested in one experiment are given, but in all observedcases penicillin potencies of fermentations with G-, S-,and W-type substrains agree with those given in table 1.

Investigations of all steps in submerged fermentationthrough small tank fermentors revealed the same pat-tern. Whenever white or sectoring colonies appearedin platings of a culture to be used, the penicillin poten-cies of the next stage and all subsequent stages were low.For further studies, a series of 10 LCS agar slants

was made from the same soil tube of strain A. After 7days' growth these slants were used to grow small tankfermentor inocula. At the same time they were platedout in serial dilutions on LCS agar and also used toinoculate shake-flask fermentations. The platings wereexamined after 10 days' growth and the fermentationswere assayed for penicillin potency after 6, 7, and 8days. The same tests were also run on the tank fer-mentor inocula when they were prepared. Results ofthese experiments are given in table 2. In this table,the slants are divided into groups based on the stage ofappearance of W- or S-type colonies. In group I thesecolony types did not appear in either the slants or inplatings of the tank inocula. In group II they did notappear in the slants but appeared to some extent inplatings of the tank inocula. Group III is composed ofone slant which showed a small number of W-type inthe slant population and a large number in the tankfermentor inoculum. These results indicate that theW- and S-type colonies appear at random in the cul-tures, at least on solid media. In all groups, penicillinpotencies agreed with that expected on the basis of thestage of appearance of W- and S-type colonies in plat-ings. Similar results were obtained when small fermen-tation tanks were inoculated with the tank inoculagiven in table 2. Group I inocula produced tank fermen-tations with high-potency broths; those inoculatedwith group II or group III inocula gave low-potencybroths. Platings of growth from tanks inoculated with

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TABLE 2. Random appearance of W-type and S-typecolonies in a pure culture of strain At

Tank FermentorLactose-Cornsteep Inoculum Grown from

Agar Slant Lactose-CornsteepAgar Slant Spores

Slant No.* W- or S-Shake- W-or S-t3Tpe Shake- typeflask colonies in flask -Coloniesyil opating yed in platingyield of slantt yield of

inoculumt

Oxford Per cent Oxford Per centunits/ml units/mi

Group 1. (No W- or S-type colonies)3 3120 0 2800 05 2790 0 2825 08 2610 0 2405 0

Group 2. (No W- or S-type colonies inslants but in platingsof tank inocula)1 2800 0 1970 27 2690 0 1730 110 2750 0 1880 1

Group 3. (Small num-bers, of W- type inslant and large no. intank fermentor inoc-ulum)4 1750 2 720 28

* All slants were inoculated at the same time from the samesoil tube of Strain A.

t W-type = white colonies; S-type = white-sectored, dark-green, colony type.

group I cultures occasionally showed a large percentageof W- and S-type colonies, however, usually only a fewwere found and these appeared late in the fermentationcycle. Tanks inoculated with group II or III alwaysshowed a large percentage of W- and S-type colonies,and these were found early in tank fermentations. Allthree colony types were isolated at some stage of thefermentation from all small tank fermentors tested.These isolates, according to strain type, all gave theexpected penicillin potencies when tested in shake flaskfermentations. When tank inoculum which showed novariants in plating tests was used, W- and S-typecolonies appeared relatively late in the tank fermenta-tion. Such tanks were always of high penicillin potency.When the variants appeared early, penicillin potencieswere low. The high-penicillin-yielding segment of thepopulation was still present in the mycelia of these low-potency tanks, and strains producing yields as high asthose of the original parent strain could be reisolatedfrom them by routine plating procedures. The W- andS-type variants did not produce substances which de-stroy penicillin already produced in the fermentation,nor any material which inhibited penicillin productionof the G-type strain in the first place. This was shown

by adding Seitz-filtered broths from W-type strainfermentations at intervals during G-type fermenta-tions, and also to completed G-type fermentation brothswhich were then allowed to stand for 16 hr. Neithertreatment reduced the yields of the G-type fermenta-tions beyond that which could be accounted for bydilution.

Strains apparently identical with the original in peni-cillin-producing abilities and morphology could easilybe reobtained from rundown cultures by serial single-colony reisolation using shake-flask cultures, slants, orsoils as starting points. All of the rundown strains de-rived from W50-935 examined by us had large percent-ages of W- and S-type variants in their populations. Inserial reisolation procedures a suitable source of therundown culture was plated on LCS agar. From thisplating a number of well-isolated typical G-typecolonies were inoculated onto LCS agar slants, using amass inoculum prepared by macerating entire coloniesin separate tubes of 0.01 per cent Wetanol solution.After 7 days' growth, these slants were plated again onLCS agar and G-type colonies picked to slants againafter 7 days. This procedure was repeated a third time.At each of the three platings, the slants were also testedfor penicillin production in shake-flask fermentations.The results of this reisolation procedure on a numberof W50-935 derivative strains are given in table 3.Note that culture E-2 was apparently pure G-typeafter the second reisolation, but the W-type variantreappeared in the third reisolation. This does not occuroften, provided growth on the platings and slants is notover 9 days old when used or transferred. This techniquehas been used on rundown or impure strains of Strep-tomyces as well as other lines of Penicillium, and strainsidentical in appearance and yields with the originalisolate are readily reobtained. Foster et al. (1943) statethat large numbers of transfers are conducive to peni-cillin rundown and recommend an absolute minimumof transfers between the soil tube and ultimate use ofthe culture in production. They also found that run-down was accompanied by sectoring and white vari-ants. However, we find that when carried out in

TABLE 3. Response of rundown cultures to serial reisolation

After After 1st After 2nd After 3rdPeni- Rundown Reisolation Reisolation Reisolationcillin

StanYield W- w- w- wStrain Beifeor Peni- type Peni- type Peni- type Peni- type

Run- Peni-n andn

and .-and -ili anddown i tllin cl S_type cilli cillin S-ype

yieldC00olo- oo yedColo-nies nies nies nies

Oxford Oxford o Oxford on Oxford Oxfordunits/ml units/mi units/ml 0 units/ml units/mli

B 2650 1250 30 2018 <0.1 2680 0 2500 0C 2600 1990 23 2730 0 2510 0 2670 0D 2500 1860 45 2560 0 2520 0 2570 0

E-1 2840 1900 38 2600 <0.1 2550 0 2550 0E-2 2840 1900 38 2680 <1.0 2660 0 2130 4

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conjunction with a selective procedure such as theabove, most of the cultures are just as potent penicillinproducers as the original isolate after as many as 10transfers.

Heterokaryon Formation Between Different PenicilliumStrains and the Effect on Penicillin Yields

A study of possible causes of rundown in Penicilliumheterokaryons was made by testing artificially synthe-sized heterokaryons in shake flasks. Shake-flask fermen-tations were inoculated with mass inocula preparedfrom slants of strains A, H, 2158, W50-935, and White# 3. All of these slants with the exception of W50-935had been established from single spores. Fermentationswere run simultaneously on each strain separately andin all possible combinations with the other single strains.These fermentations were assayed for penicillin poten-cies and plated for colony types. Results of this experi-ment are given in table 4. Such a technique allowshomokaryons and the heterokaryon type formed fromthe two homokaryons to exert their effects during thefermentation. There is little doubt that the heterokar-yon is formed under these circumstances, since allcombinations in which a white strain is mixed witha green strain give a very high percentage of white-sectoring G-type colonies and comparatively low per-

centages of the presumably homokaryotic W- andG-types of nonsectoring colonies. Sectoring coloniesdo not appear to any appreciable extent in any of thefermentations where pure W-type or pure G-type

strains were the only inocula. The fact that the greatmajority of colonies are sectored also shows that theheterokaryon is balanced (or stable) in liquid shake-flask medium, at least throughout the greater part ofthe fermentation, and that it breaks down on the solidmedium used as indicated by sectoring.Attempts to synthesize heterokaryons of the same

strain combinations were made, using the microscopeslide-culture method of Lindegren and Andrews (1945).However, these attempts were unsuccessful. All com-

binations, and even mycelia of the same strain, were

most reluctant to form anastomoses on the agar-slideculture. Possibly the same factors responsibility forheterokaryon breakdown and sectoring are also respon-

sible for the scarcity of anastomoses on solid mediumin the slide-culture tests.Another approach to this same problem was more

successful. Single hyphal tips were isolated from thewhite sectors and from the green portions of a numberof S-type colonies obtained from the platings given intable 4. In all cases, the hyphal tips taken from whitesectors gave rise only to W-type strains which gave poor

penicillin yields. Most of the tips isolated from the green

portion of S-type colonies produced S-type cultureswhich gave low to intermediate penicillin yields. How-ever, a few cultures established in this manner were ap-

parently pure G-type and gave high penicillin yields.The fact that a single hyphal strand gave rise to a

colony which produced spores of both parental types isbelieved to be strong evidence that the strain was

TABLE 4. Effect of W-type strains on colonial morphology and penicillin production of other strains

6th Day of 7th Day of 8th Day of Peak Assay andFermentation Fermentation Fermentation Highest Per Centof W-Type Colonies

W-and W- and W-and W-andYield S-type* Yield S-type Yield S-type Yield S-type

colonies colonies colonies colonies

Oxford o% Oxford e Oxford % Oxford %units/ml units/ml o units/ml units/ml

W-type strain #3 600 100 W 545 100 W 670 100 W 670 100 WStrain H (G-type strain)* 2640 O W 3250 0 W 3100 0 W 3250 0 W

OS <0.1S <1 S <1.OSW50-935 (parent strain) 640 8 W 750 11 W 620 11 W 750 11 W

70S 67 S 75 S 75 SStrain X (from different line than W50-935) 1500 0 W 1920 0 W 1740 0 W 1920 0 W

0S 0 S 0 S 0 SW-type 3 + strain H 1320 8 W 1340 3 W 1220 6 W 1340 3 W

50 S 77 S 72 S 77 SW-type #3 + W50-935 700 10W 620 5 W 840 6 W 840 6 W

68S 68 S 70 S 70 SW-type 3 + strain X 880 20 W 1180 5 W 1200 8 W 1200 8 W

20 S 15 S 13 S 13 SStrain H + W50-935 1630 OW 1380 6 W 1200 - 1630 6 W

25S 68 S 68 SStrain H + strain X 1590 OW 2120 0 W 2080 0 W 2120 0 W

0 S <0.1 S <0.1 S 0.1 SW50-935 + strain X 1100 5W 1080 3 W 900 3 W 1100 3 W

40S 54 S 60 S 60 S* G = green-type; S = sectored-type; and W = white-type.

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TABLE 5. Penicillin yields and population patterns of culturesestablished from single hyphal tips taken from white and green

sectors of the same S-type colony

Source and Isolate Shake-Flask Population PattemYield Pplto atr

Oxfordunits/ml

White sector of S-typecolony

1-S-W1 .................. 669 100% W-type1-S-W4 .................. 687 100% W-type

Green sector of S-typecolony

1-S-G1 ................... 1188 W-, S-, and G-types

1-S-G2 ................... 930 W-, S-, and G-types

1-S-G6 ................... 2553 100% G-type

heterokaryotic. The results of this experiment are givenin table 5. Only results for tests of colonies isolatedfrom the W-type # 3 x strain A fermentation are given.The same tests were made for several other combina-tions and essentially the same results were obtained.The fact that the majority of colonies are S-type in themixed strain experiments (table 4), and that the green-

type growth in these sectoring colonies will yield bothpure G-type, high-penicillin-producing strains and low-yielding S-type strains indicates that heterokaryonsare probably fairly stable in liquid medium, but thatthey break down immediately on plating on solidmedium.

Origin of the W- and S-Type ColoniesMost Penicillium species which have been studied

have been found to readily form heterokaryons (Baker,1944; Pontecorvo and Gemmell, 1944a, 1944b; Linde-gren and Andrews, 1945; Jinks, 1952a, 1952b). Jinksin his studies also found that balanced Penicilliumheterokaryons often break up into the componentstrains when they are removed to artificial media or

unnatural growth conditions. Our observations on theappearance of W- and S-type variants suggested thatheterokaryosis was the main factor involved here inpenicillin rundown or physiological degeneration.There are several mechanisms which could lead to

heterokaryosis in these strains. In one such mechanismW-type-producing nuclei, arising by mutation in themycelium, and having a selective growth advantage inthe culture, would rapidly increase in relative number.Hyphal anastomoses would spread the mutant nucleithroughout the mycelia. As their number increasedrelative to the normal type, a rundown culture wouldresult, especially if they contribute little to penicillinproduction and use much more nutritive material forgrowth than does the normal strain.A second possibility is that the original isolates of the

strain may be composed of more than one genotype.

This heterogenic condition may have arisen fromheterokaryotic mycelial fragments, polynucleate spores,or spores having heterozygous diploid nuclei (Ponte-corvo and Sermonti, 1954). In any of the above situa-tions, both W- and S-type genotypes would have to bepresent. In such a case, the W-type component wouldnecessarily have little effect on the penicillin producingability of the strain as long as it was in the balancedheterokaryotic state (or if in the same nucleus it wasrecessive to the G-type). Such a condition could be evenmore desirable than a pure strain due to hybrid vigoror heterosis contributed by the dual system (Beadleand Coonradt, 1944; Baker, 1944; Pontecorvo, 1946;Jinks, 1952a, 1952b). It is possible that with certainnuclear ratios, and under certain conditions, the hetero-karyon could be expected to have the high penicillinproducing ability of the G-type strain and the highgrowth rate of the W-type strain. However, in a mix-ture of homokaryotic (W- and G-types) and hetero-karyotic (S-type) strains, the rapidly growing W-typewould have a detrimental effect on the penicillin pro-ducing abilities of the heterokaryotic strain or theG-type strain. Thus, if for any reason the nucleardivision rates should change in favor of the W-nucleartype so as to allow it to segregate from the heterokar-yon, it would sporulate, pure W-type substrains woulddevelop from those spores containing only W-typenuclei and rundown would result.

Several factors indicate that the second mechanism,or possibly a combination of both mechanisms, is themore likely cause of rundown in these strains. The whitesectors in S-type colonies always appear on solid mediaat approximately the same time in individual experi-ments, and they usually appear in relatively large num-bers. Very seldom do they appear before the 9th or 10thday of growth. If the sectors arise as the result of muta-tions one would expect them to make random appear-ances during this time. More sectors would be expectedin the later stages of growth when the nuclei are morenumerous, but some would be expected to appear asearly as the second or third day and on all days there-after unless some unusual selective factors are operative.Instead, the white sectors seem to make their appear-ance in response to an unknown stimulus occurring ata definite time during clone growth. Moreover, thesectors are nearly always white. Only occasionally weresingle, dark-green or light-green sectors observed. Somemedium change, such as an accumulation of metabolicproducts or the exhaustion of some nutrient needed formaximum growth of the G-type component, could easilybe the causative factor. This is suggested since sectoringcan usually be prevented, and always retarded, bytransferring colonies to fresh media at 7-day intervals.This behavior would be expected of a balanced hetero-karyon in which the W-type nuclear component had aslower growth rate on fresh medium than either the

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heterokaryon or the G-type nuclear component. Underthese conditions, mycelia containing only W-typenuclei probably would not develop. If the W-typenuclei could multiply at a faster rate on old media, orif multiplication of G-type nuclei slowed down on it,production of W-type would exceed that of the G-type.When this occurred, the heterokaryon would be out ofbalance. More and more of the mycelial strands wouldbecome homokaryotic for W-type, and white sectorswould develop (see Jinks 1952a, 1952b for theory).While growth rates have not been measured by precisemethods, it is obvious by inspection that the whitecolonies and sectors grow at a faster rate than G-typecolonies. The white sectors developing in G-typecolonies are fan-shaped and Pontecorvo (1946) offersconclusive evidence that mycelia in fan-shaped sectorshave a faster growth rate than that of the parent colony.Also white colonies are usually considerably larger thanthe G-type colonies, and they produce visibly moremycelia in shake flasks than the G-type strains. Occa-sional G-type cultures have been obtained from theserial reisolation procedures which sector very infre-quently, even when they are incubated for 30 days.These cultures have never shown any evidence of run-down. All of these factors indicate that sectors andwhite colonies arise by breakdown of an establishedheterokaryon, regardless of the origin of the two nucleartypes involved.A number of factors indicate that the W-type nuclei

could have arisen in, or prior to, the W50-935 strain.Low-yielding W-type and S-type strains and high-yielding G-type strains can be isolated in considerablenumbers from platings of W50-935. Microscopic exami-nation of spores of a number of the ancestral strainsindicates the possibility that at least some of the sporesare multinucleate. Pontecorvo and Sermonti (1954),using strains derived from Q-176 (an ancestral strainof W50-935), have found that some of the spores of thisstrain are diploid. Either or both of these conditions,that is, multinucleate spores having nuclei of two ormore different types and diploids having two differentsets of chromosomes, can produce polymorphous strains.Regardless of the origin of the mixture of strains orwhether they are perpetuated by a heterozygousdiploid, polynuclear spores, or by a series of highlymutable genes, the experiments demonstrate howheterokaryosis and heterokaryon breakdown operateto bring apout penicillin rundown. They show as wellthe value of serial reisolation in controlling or eliminat-ing rundown.Many of the experiments reported here have been

performed with several strains of Streptomyces withthe same results. For example, heterokaryon breakdownhas been observed to immediately precede rundown inseveral different tetracycline-producing strains. In

these, however, it is not clear whether stable hetero-karyons are poorer producers than pure superiorstrains, or whether rundown only occurs after segrega-tion of the low-yielding component. Much the samesituation appears to exist in Streptomyces as in Peni-cillium, and for the same reasons.

ACKNOWLEDGMENTS

The authors wish to express appreciation to Dr. A.B. Hatch and Mr. W. McGhee of Bristol LaboratoriesDepartment of Fermentation Development for the tankfermentor runs, and for making periodic samples ofthese fermentations available to us. We also wish tothank Miss E. Lively of Bristol Laboratories Depart-ment of Microbiology Research for making most of thesingle-spore isolations, and Dr. A. Gourevitch of thesame department for many valuable suggestions andcriticizing the manuscript.

SUMMARYStrain rundown and culture change has been an ever-

present problem in industrial fermentations. Studies onthese phenomena occurring in Penicillium chrysogenumstrains derived from Wisconsin 50-935 are reportedhere.Rundown was found to be caused here by the appear-

ance of a low-penicillin-producing white strain in theparent culture. This white derivative, which was verystable, readily formed heterokaryons with pure high-penicillin-producing strains of the green type, and inthe balanced heterokaryotic state it did not appear todepress penicillin yields. However, under certain con-ditions the heterokaryons broke down and the purewhite strain segregated out. After this a mixed popula-tion developed containing pure white and green clonesas well as the heterokaryon. In such cultures, penicillinyields and growth of the green strain were rapidly anddrastically depressed.Using white and green substrains derived from the

same parent cultures, artificial heterokaryons weresynthesized, subjected to the various cultural conditionsencountered in industrial fermentations, and hetero-karyon effects studied. These experiments show thatheterokaryosis and heterokaryon breakdown, followingthe occurrence of mutation or genetic segregation in astable culture, can be major causes of culture rundown.Procedures for preventing this type of rundown aregiven.

Various possibilities for the origin of the white vari-ant are discussed.Many of the same heterokaryon experiments have

been repeated with certain strains of Streptomyces withthe same type of results; therefore it is to be expectedthat rundown in Streptomyces cultures can also be dueto the same causes.

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